The present invention relates to recombinant DNA which encodes the BsoBl restriction endonuclease, as well as to related methods for over-expressing this recombinant enzyme.
Nucleases are a class of enzymes which degrade or cut single- or double-stranded DNA. Restriction endonucleases are an important class of nucleases which recognize and bind to particular sequences of nucleotides (the `recognition sequence`) along the DNA molecule. Once bound, they cleave both strands of the molecule within, or to one side of, the recognition sequence. Different restriction endonucleases recognize different recognition sequences. Over one hundred and eighty restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date.
It is thought that in nature, restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteda to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by cleaving invading foreign DNA molecules when the appropriate recognition sequence is present. The cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific endonucleases.
A second component of these bacterial protective systems are the modification methylases. These enzymes are complementary to the restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA from cleavage and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of the modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins and enzymes that they encode in greater quantities than are obtainable by conventional purification techniques. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex `libraries`, i.e., populations of clones derived by `shotgun` procedures, when they occur at frequencies as low as 10.sup.-3 to 10.sup.-4.
Type II restriction-modification systems are being cloned with increasing frequency. The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRll: Kosykh et al., Molec. Gen. Genet. 178:717-719, (1980); Hhall: Mann et al., Gene 3:97-112, (1978); Pst I Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the presence of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et aL, Nucl. Acid. Res. 12: 3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); Pvull: Blumenthal et al., J. Bacteriol. 164:501-509, (1985)).
A third approach, and one that is being used to clone a growing number of systems are now being cloned by selection for an active methylase gene. See, e.g., U.S. Pat. No. 5,200,333, and Bsu Rl: Kiss et al., Nucl. Add. Res. 13: 6403-6421, (1985)). Since restriction and modification genes are often closely linked, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRl: Szomolanyi et al., Gene 10: 219-225, (1980); Bcnl: Janulaitis et al, Gene 20:197-204 (1982); BsuRl: Kiss and Baldauf, Gene 21: 111-119, (1983); and Mspl: Walder et al., J. Biol. Chem. 258: 1235-1241, (1983)).
A fourth cloning method (the "methylase indicator" method) relies on methylation-dependent restriction systems McrA, McrBC, and Mrr (Raleigh and Wilson, Proc. Natl. Acad. Sci., USA 83: 9070-9074, (1986), Heitman and Model, Gene. 103:1-9 (1991); Kelleher and Raleigh, J. Bacteriol. 173:5220-5223, (1991)) and the dinD1::lacZ operon fusion to screen for clones that contain methylase genes. The dinD1 locus is a DNA damage inducible gene that is expressed in E. coli when the "SOS response" is triggered, as by UV treatment, mitomycin treatment, or the action of McrA, McrBC, or Mrr restriction endonucleases on methylated DNA (Kenyon and Walker, Proc. Natl. Acad. Sci. USA, 77:2819-2823, (1980), Heitman and Model, Gene, 103:1-9, (1991); Heitman and Model, J. Bacteriol. 169:3234-3250, (1989); and Piekarowicz et al. J. Bacteriol. 173:150-155, (1991), the disclosures of which are incorporated herein by reference). Strains with temperature-sensitive mutations in mcrA, mcrBC, rnrr and carrying the dinD1 ::lacZ fusion were constructed and used for the direct cloning of methylase genes into E. coli from other bacterial sources (Piekarowicz etal., Nucleic Acids Res. 19:1831-1835, (1991), the disclosure of which is incorporated herein by reference). Upon transformation of ligated genomic/vector DNA into such strains, transformants containing a gene expressing a methylase that confers sensitivity to one of the methylation-dependent restriction systems form white colonies at 42.degree. C. and blue colonies at 30.degree. C. on X-gal indicator plates as a result of methylation-dependent restriction that results in SOS DNA repair induction and .beta.-galactosidase expression. Because of close linkage between most restriction enzyme genes and the cognate methylase genes, cloning of a methylase gene in a DNA fragment of reasonable size may lead to concomitant cloning of the cognate endonuclease gene.
It would be desirable to design a method for the direct cloning of nucleases, such as the BsoBl restriction endonucleases, as an alternative method when standard approaches are either impractical or fail to yield positive results.